Regarding lipoprotein, serum contains four kinds of lipoprotein, i.e. high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) and chylomicron (CM). It has been known that diseases such as various types of hyperlipemia are derived from an increase in one or more of such lipoprotein fractions and there have been reported methods for a quantitative determination by fractionation into each lipoprotein.
Fractionation of lipoprotein has been carried out by means of an ultracentrifugal operation from ancient days. The operation therefor needs skillfulness and is conducted by installing an ultracentrifuge separately and centrifugation is carried out for several days. Therefore, it was not possible to treat many samples. Besides that, there are a method where lipoprotein is separated by electrophoresis and amount of protein is determined and a method for the assay of cholesterol for each lipoprotein by HPLC but they are lacking in processing ability and, in addition, expensive apparatus in addition to the popular automatic analysis apparatus is necessary.
Recently, in order to solve the above-mentioned problem concerning the assay of cholesterol in HDL, a fully automatic kit for the assay of cholesterol in HDL has been developed and is diffusing. The art mentioned in Japanese Patent No. 2,600,065 (Nov. 30, 1994), Japanese Patent Laid-Open No. 08/201,393 (Jan. 31, 1995) and Japanese Patent Laid-Open No. 08/131,195 (Dec. 21, 1994) use a fractionating agent together but metal used as divalent cation contained in the fractionating agent forms an insoluble precipitate with detergent which is commonly used in an full-automatic analyzing apparatus and that is accumulated in a waste liquid mechanism causing a trouble. In addition, an insoluble aggregate is formed during the reaction resulting in turbidity which affects the assayed result and causes the assay error and, further, reaction cell is polluted by the aggregate which significantly affects the assayed result for other biochemical items being assayed at the same time.
There are many cases that the reaction is completed within 10 minutes in most of the popular automatic analyzing apparatuses. Moreover, it is possible to select two-point end method, rate method, double rate method, fix time method, etc. which are known methods and, therefore, assay is possible even in a turbid state. However, such an assay in a turbid state causes an error in the assayed data if there are changes in turbidity during the reaction and there is a problem in precision. In addition, reproducibility lowers when the reaction solution becomes turbid. Therefore, there is a limitation in the sample to be assayed and it is not possible to cope with broad assaying wavelengths and various kinds of samples of patients. For example, near 340 nm (UV region), there is a disadvantage that absorbance becomes 2˜3 or more due to a turbid phenomenon by the aggregate and is sometimes out of the allowable range of the analyzer. An art of Japanese Patent Laid-Open No. 09/96,637 (Jul. 19, 1996) where no divalent cation is used is a method where anti-serum aggregating with lipoprotein is contained but even the method forms a hardly soluble antigen-antibody aggregate and, therefore, the reaction cell is polluted. Accordingly, that greatly affects the assayed data for other biochemical items assayed at the same time. Further, since turbidity in the reaction solution becomes strong, precise assay is impossible by the same reason as above for the assay of cholesterol in HDL especially by UV region.
Such an art comprises a device for assaying method and common technique for inhibiting the enzymatic reaction by the formation of complex and aggregate and the bad affection inherent to the turbidity is not solved.
With regard to assay of cholesterol in LDL (LDL-C), there are reports for an assaying method of LDL-C with an object of full automation as noted in the art of Japanese Patent Laid-Open No. 07/280,812 (Apr. 5, 1994), WO 96/29599 (Mar. 20, 1995) and Japanese Patent Laid-Open No. 09/313,200 (May 29, 1996). Like the assay of cholesterol in HDL (HDL-C), those assaying methods are on the extension line of the above-mentioned art forming aggregate and complex and, therefore, it is a matter to be solved in future how the turbidity is treated.
On the other hand, in the test and diagnosis of the lipoprotein-related diseases such as hyperlipemia, information concerning HDL-C and LDL-C only is insufficient as clinical information. The World Health Organization (WHO) classifies hyperlipemia into five types [Rinsho Kensa, Vol.40, No. 9, page 107 (1996)] in which fate of cholesterol in VLDL (VLDL-C) is stipulated as well. Thus, for example, in the type IIa among the hyperlipemia II, LDL rises while, in the type IIb, LDL and VLDL rise. In the type IV, VLDL decreases while, in the type V, VLDL rises. As such, hyperlipemia is unable to be judged by the fate of single lipoprotein. Further, in the study concerning lipoprotein metabolism of coronary artery diseases such as arteriosclerosis caused by hyperlipemia, information for VLDL-C has also become necessary. Furthermore, there is a report that the fate of remnant-like lipoprotein (RLP) is a direct trigger for arteriosclerosis [McNamara J. R., et al., Clin. Chem., 44, 1224–1232 (1998)]. Assay of not only VLDL-C but also VLDL sub-fraction such as RLP-C is being regarded as important. Anyway, there is no assaying method where an operation for the separation of VLDL-C is unnecessary and, at present, there is no way but relying upon ultracentrifugation, electrophoresis, HPLC, etc. Accordingly, that is a cause of no progress in the study in spite of its clinical significance.